Biomarkers of Plasmodium falciparum infection during pregnancy in women living in northeastern Tanzania

Abstract

In pregnant women, Plasmodium falciparum infections are an important cause of maternal morbidity as well as fetal and neonatal mortality. Erythrocytes infected by these malaria-causing parasites accumulate through adhesive interactions in placental intervillous spaces, thus evading detection in peripheral blood smears. Sequestered infected erythrocytes induce inflammation, offering the possibility of detecting inflammatory mediators in peripheral blood that could act as biomarkers of placental infection. In a longitudinal, prospective study in Tanzania, we quantified a range of different cytokines, chemokines and angiogenic factors in peripheral plasma samples, taken on multiple sequential occasions during pregnancy up to and including delivery, from P. falciparum-infected women and matched uninfected controls. The results show that during healthy, uninfected pregnancies the levels of most of the panel of molecules we measured were largely unchanged except at delivery. In women with P. falciparum, however, both comparative and longitudinal assessments consistently showed that the levels of IL-10 and IP-10 increased significantly whilst that of RANTES decreased significantly, regardless of gestational age at the time the infection was detected. ROC curve analysis indicated that a combination of increased IL-10 and IP-10 levels and decreased RANTES levels might be predictive of P. falciparum infections. In conclusion, our data suggest that host biomarkers in peripheral blood may represent useful diagnostic markers of P. falciparum infection during pregnancy, but placental histology results would need to be included to verify these findings.

Figure 1. Baseline plasma levels of several inflammatory factors and others throughout pregnancies not complicated by malaria.

Plasma levels of (A) cytokines, (B) chemokines, and (C) angiogenic factors in plasma samples from 79 uninfected pregnant women measured throughout their pregnancy. Samples were analyzed at inclusion, at antenatal visit (ANV) 2, 3 and 4 and at delivery by ELISA or CBA. The dots represents the mean value and the bars the standard deviation.

Figure 2. Plasma levels of several inflammatory and other factors in pregnant women with or without P. falciparum infection.

Plasma levels of selected factors were measured in 42 P. falciparum infected women, who were infected once at single time points during pregnancy, and matched to 79 uninfected controls. The factors were measured using ELISA or CBA. The boxes represent the values between 25% and 75% quartile and the line indicates the median. The whiskers indicate the 10% and 90% percentiles. The p-values were determined by non-parametric Mann-Whitney U test.

Figure 3. Longitudinal assessment of IL-10 and IP-10 during pregnancy.

All women that were infected during their pregnancy were grouped according to the time point when the women were infected. For each group of infected women, samples from the negative control women at the same occasion were grouped together in the same graph. The three antenatal visits (ANV) were at gestational ages 26 (ANV2), 30 (ANV3), and 36 (ANV4). At inclusion: uninfected (n = 24), infected (n = 13); at ANV2: uninfected (n = 13), infected (n = 7); at ANV3: uninfected (n = 15), infected (n = 8); at ANV4: uninfected (n = 13), infected (n = 7); at Delivery: uninfected (n = 14), infected (n = 7). The red lines illustrate mean values for the infected women and the blue line illustrates the mean values for the uninfected control women. The statistical significance of differences in the concentrations between infected and uninfected women at the different time-points is illustrated.

Figure 4. Comparison of inflammatory factors in peripheral plasma from P. falciparum infected and uninfected women stratified by gravidity status.

Plasma levels of (A) MCP-1 (B) RANTES, (C) IP-10 and (D) IL-10 according to gravidity of the women. Infected primigravidae (n = 12), uninfected primigravidae (n = 22), infected secundigravidae (n = 17), uninfected secundigravidae (n = 33), infected multigravidae (n = 13) and uninfected multigravidae (n = 25). The boxes represent the values between 25% and 75% quartile and the line indicates the median. The whiskers indicate the 10% and 90% percentiles. P-values were determined by non-parametric Mann-Whitney U test.

Figure 5. Assessment of biomarkers in predicting P. falciparum infection during pregnancy.

Receiver operating curve (ROC) for the multiple logistic regression model, with IL-10, IP-10 and RANTES as predictors. The area under the ROC curve (AUC) with 95% confidence interval is 0.83 (0.75, 0.91).